DEAE-Sepharose column

In order to remove the residual PME the AE fraction was further fractionated on Mono S cation exchange column. Unlike the DEAE-Sepharose column, where PME elutes after the AE activity, the order of elution was reversed on the Mono S column (Fig. 1). The last step in the purification was a gel filtration (Sephacryl S-200) column. 

Upon purification of the XDH from C. purinolyticum, a separate Se-labeled peak appeared eluting from a DEAE sepharose column. This second peak also appeared to contain a flavin based on UV-visible spectrum. This peak did not use xanthine as a substrate for the reduction of artificial electron acceptors (2,6 dichlor-oindophenol, DCIP), and based on this altered specificity this fraction was further studied. Subsequent purification and analysis showed the enzyme complex consisted of four subunits, and contained molybdenum, iron selenium, and FAD. The most unique property of this enzyme lies in its substrate specificity. Purine, hypoxanthine (6-OH purine), and 2-OH purine were all found to serve as reductants in the presence of DCIP, yet xanthine was not a substrate at any concentration tested. The enzyme was named purine hydroxylase to differentiate it from similar enzymes that use xanthine as a substrate. To date, this is the only enzyme in the molybdenum hydroxylase family (including aldehyde oxidoreductases) that does not hydroxylate the 8-position of the purine ring. This unique substrate specificity, coupled with the studies of Andreesen on purine fermentation pathways, suggests that xanthine is the key intermediate that is broken down in a selenium-dependent purine fermentation pathway. ... 

Fractionated by passing through a DEAE-sepharose column in 10mM phosphate buffer, pH 5.8... 

Figure 4. DEAE-sepharose column chromatography of HWM endoglu-canase. (A) Peak having major endoglucanase activity and (B), (C), (D) peaks having lower amounts of endoglucanase activity.

Figure 6. DEAE-sepharose column chromatography of cellobiohydrolase. (-O-) Relative protein concentration, (- -) relative activity with respect to Avicel.

Endoglucanase and cellobiohydrolase enzyme fractions, obtained from DEAE-Sepharose column chromatography of crude enzyme from in-house-grown T. reesei culture filtrates, were examined for activity on cellulose. As expected, cellobiohydrolase action resulted in cellobiose formation while cellotriose as well as cellobiose was formed by the action of endoglucanase. In both cases, the formation of glucose was minimal. Examination of the combined activity of these components on celluloses, showed a certain degree of synergism does exist. 

POase is usually purchased although it is simple and fast to purify (Tijssen and Kurstak, 1984) decreasing the cost sometimes more than 10 times. This method starts with a low priced crude extract (RZ of 1.0) that is dissolved in 2.5 mM sodium phosphate buffer (pH 8.0) and passed over a DEAE-Sepharose column, previously equilibrated with the same buffer (up to 5 mg of protein per ml of gel). Impurities and less active acidic isozymes are retained whereas pure POase passes directly. 

If the MAb is used for the preparation of a clinical IT, an additional chromatographic purification is performed on a DEAE-Sepharose column equilibrated with PBS to remove the murine DNA and bacterial endotoxin contaminating the MAb. 

The combined fractions from the DEAE Sepharose column (450 mg, 300 ml, A490/A280 = 0.2), are loaded direcfly onto a colunrn (2.5 x 25 cm) of hydroxyapatite (high resolution, Behring Diagnostics). The adsorbed proteins are eluted with a 2100 ml gradient from 0 to 500 mM potassium phosphate in 50 mM Tris/HCl,... 

Actually, Harbury s approach exploits DNA templating in a different way. Strictly speaking, the library is not synthesized by templated reactions. DNA templates are not used to bring reactants in proximity to boost the effective molarity instead DNA templating is the mean to spatially partition the templates into sub-pools. The advantage of this approach is mostly that chemical building blocks can be directly used without the need to couple to a DNA strand and no cleavable linkers are needed second, the chemical reactions take place on solid support (DEAE Sepharose column), therefore potentially more chemical reactions can be applied to create libraries with more diverse structures. However, so far there has been no report of library synthesis other than peptidic structures. 

Reverse transcriptase (from avian or murine RNA tumour viruses) [9068-38-6] [EC 2.7.7.49]. Purified by solubilising the virus with non-ionic detergent. Lysed virions were adsorbed on DEAE-cellulose or DEAE-Sephadex columns and the enzyme eluted with a salt gradient, then chromatographed on a phosphocellulose column and enzyme activity eluted in a salt gradient. Purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 7977 Smith Methods Enzymol 65 560 1980 see commercial catalogues for other transcriptases.]... 

Because plants present chlorophylls and carotenoids simultaneously, it may be useful to separate both groups from each other in a laboratory or preparative scale in order to avoid contamination in further purification steps, mainly when they are prepared in large amounts. Clean-up procedures using an open column packed with absorbents such as alumina, magnesia, polyethylene powder, powdered sucrose, DEAE-Sepharose, starch, cellulose, or MgO HyfloSupercel are good approaches. MgO HyfloSupercel in a proportion of 1 1 or 1 2 is the usual adsorbent. Sucrose and cellulose are interesting as they do not alter the chlorophylls, but they are tedious to work with. 

Crude polysaccharide fraction (GL-2) was prepared from the leaves of P. ginseng by hot water extraction, ethanol precipitation and dialysis, and GL-2 was fractionated by Cetavlon precipitation and weakly acidic polysaccharide fraction (GL-4) was obtained[3]. GL-4IIb2 was purified from GL-4 by DEAE-Sepharose CL-6B as described previousely [3]. In order to remove the color-materials, GL-4IIb2 was further purified by Q-Sepharose (C1 form), and the major fraction, eluted with 0.3 M NaCl, was repurifled by gel filtration on Bio-gel P-30 column to obtain purified active polysaccharide, GL-4IIb2. ... 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Figure 2. Anion exchange chromatogram of ionically bound phloem peroxidases on DEAE-Sepharose. Collected fractions were analyzed for their oxidase activity towards TMB (1), syringaldazine (2) and isopropylamine salt from / -fluoroferulic acid (3). Bo cationic peroxidases Bj and B2 anionic peroxidases. Column was equilibrated with 0.01 M phosphate buffer (pH 7.1). Fractions were eluted with a NaCl gradient (0-0.5 M) in the same buffer (0.01 M phosphate, pH 7.1).

FIGURE 11.7 Selective displacement of AVP on DEAE Sepharose Fast Elow. Column dimensions were 10 X 290mm load was 6.5 mL AVP feedstock/mL resin. The flow rate was 1 mL/min. (Reprinted with permission from Elsevier from Barnthouse, K.A. et ah, J. BiotechnoL, 66, 125, 1998. Copyright.)... 

Stipe powder of C. comatus (100 g) was extracted three times with 1 L 95% ethanol under reflux for 2 h to remove lipid, and the residue was extracted three times with 2 L distilled water for 2 h at 80 °C with intermediate centrifugation (2000 x g, 15 min). After concentrating the collected aqueous supernatants to 400 mL (reduced pressure at 40 °C), a precipitation was performed with 3 volumes of 95% ethanol. The precipitate was washed with ethanol and acetone, and then dried at 40 C, yielding crude polysaccharide material. Crude polysaccharide material was dissolved in 100 mL 0.2 M sodium phosphate buffer (pH 6.0), and after centrifugation the solution was applied to a DEAE-Sepharose CL-6B column. 

DEAE-Sepharose fast flow anion-exchange chromatography column (10 x 300 mm)... 

Chi is purified from chloroplast extracts, usually obtained from spinach leaves, by dioxane precipitation method (51) and conventional sugar column chromatography (52). For rapid and easy preparation, the method recently developed by Omata and Murata (53) is satisfactory synthetic (DEAE-) Sepharose is substituted for sugar on the column. Besides using spectroscopic criteria (52), the purity of Chi samples can be checked readily by means of silica-gel thin layer chromatography (54). Colorless contaminations in Chi... 

Materials. Male rabbits (New Zealand White) were obtained from the Department of Experimental Animals of this Institute. DEAE-Sepharose, CL-6B, was a product of Pharmacia Fine Chemicals, Uppsala, Sweden and was converted to acetate form as described previously (7). latrobeads (6RS 8060) for column chromatography were supplied from latron Laboratory, Tokyo and were washed with chloroform/methanol/5N ammonia (3 2 1, by vol.) until the solution becomes colorless. 

For fast and simple purification of RNA oligonucleotides from crude transcription reactions, we use an AKTA prime FPLC system equipped with a 50-ml superloop and three 5 ml HiTrap diethylaminoethyl (DEAE) sepharose Fast-Flow columns (GE Healthcare) connected in series. The DEAE columns are equilibrated with 3 CV of buffer A (50 mM sodium phosphate, pH 6.5, 150 mMsodium chloride, and 0.2 mMEDTA) at room temperature. Buffer B contains the same components with 2 Msodium chloride. Both buffers can be prepared in large quantities, sterile filtered and stored at 4 °C (buffer A) or room temperature (buffer B) to avoid precipitation of sodium chloride. 

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